This project will elucidate the mechanism of plasmid DNA transfer during conjugation in bacteria. Conjugation systems in bacteria are encoded by plasmids, and this project is focused on the transfer system of the IncP plasmid RK2. P plasmids encode antibiotic resistance genes and are transferrable to most gram-negative bacteria. This broad host range property is of considerable medical significance in the spread of drug resistance. In addition, P plasmids are increasingly used as genetic tools in many gram-negative bacteria. Previous work in our laboratory has focused on the events surrounding the initiation of plasmid DNA transfer and conjugal DNA synthesis. These studies have led to a model for the transfer process: 1) transfer initiation proteins recognize and bind to the transfer original (oriT) region, 2) the protein(s) nicks the DNA and associates with the 5' end of the nicked strand, 3) the nicked strand is transferred to the recipient, and 4) the plasmid-encoded DNA primase binds to the oriT region and initiates synthesis of a complementary strand in the recipient. The oriT region has been sequenced, and the major structural feature is a set of 19 bp inverted repeats. The overall goal of this project is to confirm the features of this proposed model and to determine the role of the inverted repeats and other DNA sequences in each of the biochemical steps of the transfer process. The specific aims of this proposal are to determine 1) the function of the inverted repeats and flanking sequences of the oriT region, both in oriT activity and as a site for initiation of conjugal DNA synthesis by the plasmid-encoded primase, 2) the primase recognition site in oriT, 3) the DNA strand that is nicked and transferred, and to establish the polarity of transfer, 4) the binding site for the relaxation proteins, and 5) the mechanism of recircularization of the transferred strand. These studies will use current techniques in biochemical and molecular genetics, including cloning, in vitro mutagenesis, DNA sequencing, and in vitro DNA synthesis.